Collagen 3

The migratory behavior of CAFs on a 2-dimensional substrate was assessed as previously described (44 (link)). Even though a 2-dimensional setting imposes nonphysiological constraints, it offers a reliable readout regarding the adhesive and migratory machinery of the cells (65 (link)). Briefly, CAFs originating from PDAC-bearing mice were seeded in 12.5 cm² flasks that were precoated with ECM components resembling the desmoplastic stroma (66 (link)), containing the following components: 40 μg/mL laminin (Sigma-Aldrich, Merck KGaA), 40 μg/mL fibronectin (Sigma-Aldrich, Merck KGaAy), 800 μg/mL collagen I (Corning), 12 μg/mL collagen III (Corning), and 5.4 μg/mL collagen IV (BD Biosciences). Following cell adhesion overnight at 37°C containing 5% CO₂, flasks were sealed air-tight and inserted into 37°C temperature-controlled chambers. In the chambers, cell motility was observed using an inverted microscope (Zeiss Axiovert 40, Zeiss) and recorded every other 15 minutes using the MicroCamLab 3.1 software (Bresser). From the recordings, cell contours were segmented using the Amira software (Thermo Fisher Scientific) and further processed to determine cell velocities (μm/min) from the displacement of the cell centroid over time (quantification detailed by Dieterich et al.; ref. 67 (link)). Cell translocation indicates the start-to-end net cellular displacement.

The migratory behavior of CAFs on a 2-dimensional substrate was assessed as previously described (44 (link)). Even though a 2-dimensional setting imposes nonphysiological constraints, it offers a reliable readout regarding the adhesive and migratory machinery of the cells (65 (link)). Briefly, CAFs originating from PDAC-bearing mice were seeded in 12.5 cm² flasks that were precoated with ECM components resembling the desmoplastic stroma (66 (link)), containing the following components: 40 μg/mL laminin (Sigma-Aldrich, Merck KGaA), 40 μg/mL fibronectin (Sigma-Aldrich, Merck KGaAy), 800 μg/mL collagen I (Corning), 12 μg/mL collagen III (Corning), and 5.4 μg/mL collagen IV (BD Biosciences). Following cell adhesion overnight at 37°C containing 5% CO₂, flasks were sealed air-tight and inserted into 37°C temperature-controlled chambers. In the chambers, cell motility was observed using an inverted microscope (Zeiss Axiovert 40, Zeiss) and recorded every other 15 minutes using the MicroCamLab 3.1 software (Bresser). From the recordings, cell contours were segmented using the Amira software (Thermo Fisher Scientific) and further processed to determine cell velocities (μm/min) from the displacement of the cell centroid over time (quantification detailed by Dieterich et al.; ref. 67 (link)). Cell translocation indicates the start-to-end net cellular displacement.

Cell migration was recorded in a 3D matrix using an Ibidi μ-Slide I Luer. Cells were suspended at a final density of 1 × 10⁶ cells/mL and supplemented with the desired treatment compounds at the indicated concentration. Then cells were mixed in a ratio of 1:4 with an ECM-like matrix of the following composition: RPMI (10.4 g/L), HEPES (10 mM), laminin (20 μg/mL; Sigma Aldrich), fibronectin (40 μg/μL, Becton Dickinson), collagen IV (5.4 μg/μL; Corning), collagen III (12 μg/μL, Corning), rat tail collagen I (1.600 μg/mL, R&D system) and H₂O adjusted to pH 7.4 with 1 M NaOH. A final volume of 200 μL of the matrix/NSCLC cell suspension was loaded into an Ibidi μ-Slide I Luer and let polymerize at 37°C for 3 h (with 10 ng/mL EGF (Sigma Aldrich)). After polymerization, the migration experiment started. The focal plane was set in the middle of the μ-Slide. Images were acquired in 10 min intervals for 12 h using a ZEISS microscope Axiovert 40 C (Carl Zeiss, Germany), linked to a MicroCam SP 3.1 video camera (Bresser, Germany) controlled by the MicroCamLab software (Bresser). Migration of NSCLC cells was analyzed with Amira^® software (ThermoFisher). Using a self-made JAVA program the positions of the centroids were determined for each time point. Migration speed and translocation were calculated as shown by Glaser et al. 2021 [17 (link)].